We have used 18 neutralizing monoclonal antibodies (MAbs) specific for the respiratory syncytial virus (RSV) fusion (F) glycoprotein to construct operational, topologic, and functional epitope maps of this F glycoprotein. The MAbs define 16 neutralization epitopes which are organized into three non-overlapping antigenic sites (A,B,and C) and one bridge site (AB). Neutralization and fusion-inhibition tests of the MAbs indicated that antigenic sites A,AB,and C correspond to domains of the F protein that affect syncytium-formation, whereas antigenic site B is more distant from the fusion site. This interpretation is supported by two additional observations. First, MAb-resistant variants selected with site B MAbs produce normal plaques, whereas those selected with MAbs binding epitopes in sites A and AB produce pinpoint plaques. Second, epitopes in antigenic sites A,AB,and C (which correspond to domains close to the fusion site) were less variable among RSV clinical isolates than were epitopes in antigenic site B. Sequence analysis of the F genes of antibody-resistant mutants is currently in progress. This information will allow us to identify amino acid residues which are involved in binding neutralizing antibodies and which directly affect syncytium-formation.